Urban Birds as Antimicrobial Resistance Sentinels: White Storks Showed Higher Multidrug-Resistant Escherichia coli Levels Than Seagulls in Central Spain.

The presence of AMR bacteria in the human-animal-environmental interface is a clear example of the One Health medicine. Several studies evidence the presence of resistant bacteria in wildlife, which can be used as a good indicator of anthropization level on the ecosystem. The fast increase in AMR in the environment in the last decade has been led by several factors as globalization and migration. Migratory birds can travel hundreds of kilometers and disseminate pathogens and AMR through different regions or even continents. The aim of this study was to compare the level of AMR in three migratory bird species: Ciconia ciconia, Larus fuscus and Chroicocephalus ridibundus. For this purpose, commensal Escherichia coli has been considered a useful indicator for AMR studies. After E. coli isolation from individual cloacal swabs, antimicrobial susceptibility tests were performed by the disk-diffusion method, including 17 different antibiotics. A total of 63.2% of gulls had resistant strains, in contrast to 31.6% of white storks. Out of all the resistant strains, 38.9% were considered multi-drug resistant (50% of white storks and 30% of seagulls). The antibiotic classes with the highest rate of AMR were betalactamics, quinolones and tetracyclines, the most commonly used antibiotic in human and veterinary medicine in Spain.

Tuberculosis vaccination sequence effect on protection in wild boar.

The Eurasian wild boar (Sus scrofa) is a reservoir for tuberculosis (TB) in which vaccination is a valuable tool for control. We evaluated the protection and immune response achieved by homologous and heterologous regimes administering BCG and heat-inactivated Mycobacterium bovis (IV). Twenty-one wild boar piglets were randomly allocated in five groups: Control, homologous BCG, homologous IV, heterologous IV-BCG, heterologous BCG-IV. Significant 67% and 66% total lesion score reductions were detected in homologous IV (IVx2) and heterologous IV-BCG groups when compared with Control group (F4,16 = 6.393, p = 0.003; Bonferroni Control vs IVx2 p = 0.026, Tukey Control vs IV-BCG p = 0.021). No significant differences were found for homologous BCG (although a 48% reduction in total lesion score was recorded) and BCG-IV (3% reduction). Heterologous regimes did not improve protection over homologous regimes in the wild boar model and showed variable results from no protection to similar protection as homologous regimes. Therefore, homologous regimes remain the best option to vaccinate wild boar against TB. Moreover, vaccine sequence dramatically influenced the outcome underlining the relevance of studying the effects of prior sensitization in the outcome of vaccination.

Impact of piglet oral vaccination against tuberculosis in endemic free-ranging wild boar populations.

The Eurasian wild boar (Sus scrofa) is the main wild reservoir of the Mycobacterium tuberculosis complex in Mediterranean woodlands and a key risk factor for cattle tuberculosis (TB) breakdowns. Wild boar vaccination therefore has the potential to be a valuable tool for TB control. We tested two orally delivered vaccines, heat-inactivated Mycobacterium bovis (IV) and BCG, in four sites (two per vaccine type: one Managed and one Natural or unmanaged) during four years. TB was also monitored in 15 unvaccinated sites (spatial control), as well as in all sites from one year prior to intervention (temporal control). The rationale is that by vaccinating 2-6 month old wild boar piglets we can reduce disease at the population level during the study period. This is achievable due to the fast turnover of wild boar populations. Vaccine baits were deployed using selective piglet feeders and this method proved highly successful with uptake rates of 50 to 74% in Natural sites and 89 to 92% in Managed sites. This is relevant for the potential delivery of vaccines to control other diseases, too. Local wild boar TB prevalence at the beginning of the study was already high ranging from 50 to 100%. TB prevalence increased in unvaccinated sites (6%), while a significant decline occurred in the Managed IV site (34%). Changes recorded in the remaining sites were not significant. The short-term impact of vaccination observed in the field was complemented by mathematical modelling, representative of the field system, which examined the long-term impact and showed that vaccination of piglets reduced prevalence and increased abundance at the population level. We conclude that IV could become part of integrated TB control schemes, although its application must be tailored for each specific site.

Immunohistochemical detection of pro-inflammatory and anti-inflammatory cytokines in granulomas in cattle with natural Mycobacterium bovis infection.

Cellular immune response was evaluated in lymph nodes and lung with different granulomatous lesions from cattle naturally infected with Mycobacterium bovis. For this purpose, we assessed pro-inflammatory and anti-inflammatory cytokines by immunohistochemical assays. Immunoreaction was observed for all the cytokines analyzed. Fourteen animals displayed advanced stage IV granulomas, with intense immunoreactivity to IFN-γ and TGF-ß in areas of caseous necrosis, macrophages and lymphocytes. Seven animals showed stage III granuloma, with high immunoreactivity to IFN-γ (average of 44.5% immunoreactive cells) and moderate to TNF-α and to the anti-inflammatory cytokines IL-10 and TGF-ß, in relation to the proliferation of fibroblasts in granuloma periphery We found satellite stage I granulomas in 4 bovines and stage II granulomas in 2 bovines, which exhibited low immunostaining response (-13%). Cytokine expression in stage III and IV granulomas was significant, with predominance of immunoreactivity to IFN-γ, thus suggesting a strong, longstanding local immune response mediated by macrophages and epithelioid cells. In addition, these two stages displayed lower reactivity to IL-10; which suggests a deficit of anti-inflammatory cytokines, suppressed immunity and persistence of the infection. High expression of TGF-ß could indicate a chronic process with greater tissue damage and fibrosis. Numerous bacilli observed in necrotic areas in stage III and IV granulomas with low expression of IL-1ß suggest failure in the immune response with bacterial multiplication. In this study, evidence of in situ presence of cytokines demonstrates these cytokines are involved in the development and evolution of bovine tuberculosis granulomas.

Significance and integration of molecular diagnostics in the framework of veterinary practice.

The field of molecular diagnostics in veterinary practice is rapidly evolving. An array of molecular techniques of different complexity is available to facilitate the fast and specific diagnosis of animal diseases. The choice for the adequate technique is dependent on the mission and attributions of the laboratory and requires both a knowledge of the molecular biology basis and of its limitations. The ability to quickly detect pathogens and their characteristics would allow for precise decision-making and target measures such as prophylaxis, appropriate therapy, and biosafety plans to control disease outbreaks. In practice, taking benefit of the huge amount of data that can be obtained using molecular techniques highlights the need of collaboration between veterinarians in the laboratory and practitioners.

[Influenza: a four-year evolution of the pandemic. Prof. Alejandro Posadas National Hospital, Argentina]. / Influenza: evolución a cuatro años de la pandemia. Hospital Nacional Profesor Alejandro Posadas, Argentina.

As from January to August 2013, epidemiological weeks 1-35 (EW), Influenza incidence, case characteristics, types and subtypes of circulating influenza virus in the Nacional Profesor Alejandro Posadas Hospital were studied, and were compared to incidences during 2009-2012. From late May to the end of August 2013 (EW18-35), an increase was observed in the proportion of patients' visits for respiratory disease, influenza-like illness and hospitalizations due to pneumonia; of 207 cases diagnosed with influenza A virus, 153 were infected by H1N1pdm09, 46 by H3, and eight without subtype. The highest proportion of cases was found in children under five years of age, followed by the group 60-64. The chances of having the illness were three times greater among the group 40-64 years old compared to 15-39 or those older than 64. Mortality, which increased with age, was 7.2%, and the odds of death were six times higher among those older than 64. Vaccination rate among the cases was 11.6%. None of the fatal cases had received the vaccine. After the 2009 pandemic, the proportions of annual patients' visits decreased until 2012; in 2013, an increase of 52.0% during the winter period compared to 2012. The viral circulation started earlier in 2013 compared to previous years. FLU-A(H1N1pdm) was the predominant circulating virus in 2009 and 2013, FLU-A(H3) in 2011, FLU-A(H3) and FLU-B in both 2010 and 2012.

Influenza: evolución a cuatro años de la pandemia: Hospital Nacional Profesor Alejandro Posadas, Argentina / Influenza: A four-year evolution of the pandemic: Prof. Alejandro Posadas National Hospital, Argentina

En el Hospital Nacional Profesor Alejandro Posadas se estudiaron la incidencia de influenza, las características de casos y tipos y subtipos de virus circulantes de enero a agosto de 2013 inclusive, semanas epidemiológicas (SE) 1-35, y se compararon con los años 2009-2012. De fin de mayo a agosto inclusive de 2013 (SE 18 a 35) se observó un aumento del porcentaje de consulta por enfermedades respiratorias, enfermedad tipo influenza e internación por neumonía y se diagnosticaron 207 casos: 153 influenza A (FLU-A)(H1N1pdm09), 46 A(H3), ocho A(sin subtipificar). La mayor frecuencia fue en menores de 5 años, seguida por el grupo de 60 a 64.La chance de tener la enfermedad fue tres veces mayor en el grupo de 40-64 años versus 15-39 o > 64 años. La letalidad, que aumentó con la edad, fue de 7.2% y la chance de morir fue seis veces mayor en los > 64 años. El porcentaje de vacunación entre los casos fue11.6%. Ninguno de los fallecidos estaba vacunado. Luego de la pandemia de 2009 el porcentaje de consultas anuales disminuyó hasta 2012, con un aumento en el período invernal de 2013 de 52.0% con respecto a 2012. La circulación viral en 2013 fue más temprana que en los años anteriores. En 2009 y 2013 la mayor circulación fue FLU-A (H1N1pdm), en 2011 FLU-A(H3) y en 2010 y 2012 FLU-A(H3) y FLU-B.

As from January to August 2013, epidemiological weeks 1-35 (EW), Influenza incidence, case characteristics, types and subtypes of circulating influenza virus in the Nacional Profesor Alejandro Posadas Hospital were studied, and were compared to incidences during 2009-2012. From late May to the end of August 2013 (EW18-35), an increase was observed in the proportion of patients' visits for respiratory disease, influenza-like illness and hospitalizations due to pneumonia; of 207 cases diagnosed with influenza A virus, 153 were infected by H1N1pdm09, 46 by H3, and eight without subtype. The highest proportion of cases was found in children under five years of age, followed by the group 60-64.The chances of having the illness were three times greater among the group 40-64 years old compared to 15-39 or those older than 64. Mortality, which increased with age, was 7.2%, and the odds of death were six times higher among those older than 64. Vaccination rate among the cases was 11.6%. None of the fatal cases had received the vaccine. After the 2009 pandemic, the proportions of annual patients´ visits decreased until 2012; in 2013, an increase of 52.0% during the winter period compared to 2012. The viral circulation started earlier in 2013 compared to previous years. FLU-A(H1N1pdm) was the predominant circulating virus in 2009 and 2013, FLU-A(H3) in 2011, FLU-A(H3) and FLU-B in both 2010 and 2012.

Influenza: evolución a cuatro años de la pandemia: Hospital Nacional Profesor Alejandro Posadas, Argentina / Influenza: A four-year evolution of the pandemic: Prof. Alejandro Posadas National Hospital, Argentina

En el Hospital Nacional Profesor Alejandro Posadas se estudiaron la incidencia de influenza, las características de casos y tipos y subtipos de virus circulantes de enero a agosto de 2013 inclusive, semanas epidemiológicas (SE) 1-35, y se compararon con los años 2009-2012. De fin de mayo a agosto inclusive de 2013 (SE 18 a 35) se observó un aumento del porcentaje de consulta por enfermedades respiratorias, enfermedad tipo influenza e internación por neumonía y se diagnosticaron 207 casos: 153 influenza A (FLU-A)(H1N1pdm09), 46 A(H3), ocho A(sin subtipificar). La mayor frecuencia fue en menores de 5 años, seguida por el grupo de 60 a 64.La chance de tener la enfermedad fue tres veces mayor en el grupo de 40-64 años versus 15-39 o > 64 años. La letalidad, que aumentó con la edad, fue de 7.2% y la chance de morir fue seis veces mayor en los > 64 años. El porcentaje de vacunación entre los casos fue11.6%. Ninguno de los fallecidos estaba vacunado. Luego de la pandemia de 2009 el porcentaje de consultas anuales disminuyó hasta 2012, con un aumento en el período invernal de 2013 de 52.0% con respecto a 2012. La circulación viral en 2013 fue más temprana que en los años anteriores. En 2009 y 2013 la mayor circulación fue FLU-A (H1N1pdm), en 2011 FLU-A(H3) y en 2010 y 2012 FLU-A(H3) y FLU-B.(AU)

As from January to August 2013, epidemiological weeks 1-35 (EW), Influenza incidence, case characteristics, types and subtypes of circulating influenza virus in the Nacional Profesor Alejandro Posadas Hospital were studied, and were compared to incidences during 2009-2012. From late May to the end of August 2013 (EW18-35), an increase was observed in the proportion of patients visits for respiratory disease, influenza-like illness and hospitalizations due to pneumonia; of 207 cases diagnosed with influenza A virus, 153 were infected by H1N1pdm09, 46 by H3, and eight without subtype. The highest proportion of cases was found in children under five years of age, followed by the group 60-64.The chances of having the illness were three times greater among the group 40-64 years old compared to 15-39 or those older than 64. Mortality, which increased with age, was 7.2%, and the odds of death were six times higher among those older than 64. Vaccination rate among the cases was 11.6%. None of the fatal cases had received the vaccine. After the 2009 pandemic, the proportions of annual patients´ visits decreased until 2012; in 2013, an increase of 52.0% during the winter period compared to 2012. The viral circulation started earlier in 2013 compared to previous years. FLU-A(H1N1pdm) was the predominant circulating virus in 2009 and 2013, FLU-A(H3) in 2011, FLU-A(H3) and FLU-B in both 2010 and 2012.(AU)

Oral vaccination with heat inactivated Mycobacterium bovis activates the complement system to protect against tuberculosis.

Tuberculosis (TB) remains a pandemic affecting billions of people worldwide, thus stressing the need for new vaccines. Defining the correlates of vaccine protection is essential to achieve this goal. In this study, we used the wild boar model for mycobacterial infection and TB to characterize the protective mechanisms elicited by a new heat inactivated Mycobacterium bovis vaccine (IV). Oral vaccination with the IV resulted in significantly lower culture and lesion scores, particularly in the thorax, suggesting that the IV might provide a novel vaccine for TB control with special impact on the prevention of pulmonary disease, which is one of the limitations of current vaccines. Oral vaccination with the IV induced an adaptive antibody response and activation of the innate immune response including the complement component C3 and inflammasome. Mycobacterial DNA/RNA was not involved in inflammasome activation but increased C3 production by a still unknown mechanism. The results also suggested a protective mechanism mediated by the activation of IFN-γ producing CD8+ T cells by MHC I antigen presenting dendritic cells (DCs) in response to vaccination with the IV, without a clear role for Th1 CD4+ T cells. These results support a role for DCs in triggering the immune response to the IV through a mechanism similar to the phagocyte response to PAMPs with a central role for C3 in protection against mycobacterial infection. Higher C3 levels may allow increased opsonophagocytosis and effective bacterial clearance, while interfering with CR3-mediated opsonic and nonopsonic phagocytosis of mycobacteria, a process that could be enhanced by specific antibodies against mycobacterial proteins induced by vaccination with the IV. These results suggest that the IV acts through novel mechanisms to protect against TB in wild boar.

Strategic use of serology for the diagnosis of bovine tuberculosis after intradermal skin testing.

Diagnostic tests based on cell-mediated immunity are used in programmes for eradication of bovine tuberculosis (Mycobacterium bovis). Serological assays could be applied as ancillary methods to detect infected animals. Our objective was to evaluate two serological techniques: M. bovis Ab Test (IDEXX, USA) and Enferplex™ TB assay (Enfer, Ireland) in animals tested simultaneously with the single and comparative intradermal tests and the interferon-gamma assay. This work was performed at two stages. First, a preliminary panel of samples collected prior to intradermal tests from tuberculosis-free (n=60) and M. bovis-infected herds (n=78) was assayed, obtaining high specificity: 100% (M. bovis Ab Test) and 98.3% (Enferplex TB assay) but low sensitivity (detection of M. bovis infected animals): 23.9% (M. bovis Ab Test) and 32.6% (Enferplex TB assay). Subsequently, the use of serological techniques was further studied in two herds with M. bovis infection (n=77) using samples collected prior to, and 72 h and 15 days after PPD inoculation. The highest level of detection of infected animals for serology was achieved at 15 days post-intradermal tests taking advantage of the anamnestic effect: 70.4% and 85.2% in herd A, and 66.7% and 83.3% in herd B, using M. bovis Ab Test and Enferplex TB assay, respectively. Quantitative results (average values obtained with M. bovis Ab Test ELISA and degree of positivity obtained with Enferplex TB assay) were higher in animals showing lesions compatible with tuberculosis. No significant differences were observed in the number of confirmed infected animals detected with either serological technique.

Oral re-vaccination of Eurasian wild boar with Mycobacterium bovis BCG yields a strong protective response against challenge with a field strain.

BACKGROUND: Field vaccination trials with Mycobacterium bovis BCG, an attenuated mutant of M. bovis, are ongoing in Spain, where the Eurasian wild boar (Sus scrofa) is regarded as the main driver of animal tuberculosis (TB). The oral baiting strategy consists in deploying vaccine baits twice each summer, in order to gain access to a high proportion of wild boar piglets. The aim of this study was to assess the response of wild boar to re-vaccination with BCG and to subsequent challenge with an M. bovis field strain. RESULTS: BCG re-vaccinated wild boar showed reductions of 75.8% in lesion score and 66.9% in culture score, as compared to unvaccinated controls. Only one of nine vaccinated wild boar had a culture-confirmed lung infection, as compared to seven of eight controls. Serum antibody levels were highly variable and did not differ significantly between BCG re-vaccinated wild boar and controls. Gamma IFN levels differed significantly between BCG re-vaccinated wild boar and controls. The mRNA levels for IL-1b, C3 and MUT were significantly higher in vaccinated wild boar when compared to controls after vaccination and decreased after mycobacterial challenge. CONCLUSIONS: Oral re-vaccination of wild boar with BCG yields a strong protective response against challenge with a field strain. Moreover, re-vaccination of wild boar with BCG is not counterproductive. These findings are relevant given that re-vaccination is likely to happen under real (field) conditions.

Overview and phylogeny of Mycobacterium tuberculosis complex organisms: implications for diagnostics and legislation of bovine tuberculosis.

Members of the Mycobacterium tuberculosis complex (MTBC) cause a serious disease with similar pathology, tuberculosis; in this review, bovine tuberculosis will be considered as disease caused by any member of the MTBC in bovids. Bovine tuberculosis is responsible for significant economic loss due to costly eradication programs and trade limitations and poses a threat to both endangered and protected species as well as to public health. We here give an overview on all members of the MTBC, focusing on their isolation from different animal hosts. We also review the recent advances made in elucidating the evolutionary and phylogenetic relationships of members of the MTBC. Because the nomenclature of the MTBC is controversial, its members have been considered species, subspecies or ecotypes, this review discusses the possible implications for diagnostics and the legal consequences of naming of new species.

Assessment of an oral Mycobacterium bovis BCG vaccine and an inactivated M. bovis preparation for wild boar in terms of adverse reactions, vaccine strain survival, and uptake by nontarget species.

Wildlife vaccination is increasingly being considered as an option for tuberculosis control. We combined data from laboratory trials and an ongoing field trial to assess the risk of an oral Mycobacterium bovis BCG vaccine and a prototype heat-inactivated Mycobacterium bovis preparation for Eurasian wild boar (Sus scrofa). We studied adverse reactions, BCG survival, BCG excretion, and bait uptake by nontarget species. No adverse reactions were observed after administration of BCG (n = 27) or inactivated M. bovis (n = 21). BCG was not found at necropsy (175 to 300 days postvaccination [n = 27]). No BCG excretion was detected in fecal samples (n = 162) or in urine or nasal, oral, or fecal swab samples at 258 days postvaccination (n = 29). In the field, we found no evidence of loss of BCG viability in baits collected after 36 h (temperature range, 11°C to 41°C). Camera trapping showed that wild boar (39%) and birds (56%) were the most frequent visitors to bait stations (selective feeders). Wild boar activity patterns were nocturnal, while diurnal activities were recorded for all bird species. We found large proportions of chewed capsules (29%) (likely ingestion of the vaccine) and lost baits (39%) (presumably consumed), and the proportion of chewed capsules showed a positive correlation with the presence of wild boar. Both results suggest proper bait consumption (68%). These results indicate that BCG vaccination in wild boar is safe and that, while bait consumption by other species is possible, this can be minimized by using selective cages and strict timing of bait deployment.

Influenza: evolución a cuatro años de la pandemia. Hospital Nacional Profesor Alejandro Posadas, Argentina. / [Influenza: a four-year evolution of the pandemic. Prof. Alejandro Posadas National Hospital, Argentina].

As from January to August 2013, epidemiological weeks 1-35 (EW), Influenza incidence, case characteristics, types and subtypes of circulating influenza virus in the Nacional Profesor Alejandro Posadas Hospital were studied, and were compared to incidences during 2009-2012. From late May to the end of August 2013 (EW18-35), an increase was observed in the proportion of patients visits for respiratory disease, influenza-like illness and hospitalizations due to pneumonia; of 207 cases diagnosed with influenza A virus, 153 were infected by H1N1pdm09, 46 by H3, and eight without subtype. The highest proportion of cases was found in children under five years of age, followed by the group 60-64. The chances of having the illness were three times greater among the group 40-64 years old compared to 15-39 or those older than 64. Mortality, which increased with age, was 7.2

, and the odds of death were six times higher among those older than 64. Vaccination rate among the cases was 11.6

. None of the fatal cases had received the vaccine. After the 2009 pandemic, the proportions of annual patients visits decreased until 2012; in 2013, an increase of 52.0

during the winter period compared to 2012. The viral circulation started earlier in 2013 compared to previous years. FLU-A(H1N1pdm) was the predominant circulating virus in 2009 and 2013, FLU-A(H3) in 2011, FLU-A(H3) and FLU-B in both 2010 and 2012.

Splitting of a prevalent Mycobacterium bovis spoligotype by variable-number tandem-repeat typing reveals high heterogeneity in an evolving clonal group.

Mycobacterium bovis populations in countries with persistent bovine tuberculosis usually show a prevalent spoligotype with a wide geographical distribution. This study applied mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) typing to a random panel of 115 M. bovis isolates that are representative of the most frequent spoligotype in the Iberian Peninsula, SB0121. VNTR typing targeted nine loci: ETR-A (alias VNTR2165), ETR-B (VNTR2461), ETR-D (MIRU4, VNTR580), ETR-E (MIRU31, VNTR3192), MIRU26 (VNTR2996), QUB11a (VNTR2163a), QUB11b (VNTR2163b), QUB26 (VNTR4052), and QUB3232 (VNTR3232). We found a high degree of diversity among the studied isolates (discriminatory index [D] = 0.9856), which were split into 65 different MIRU-VNTR types. An alternative short-format MIRU-VNTR typing targeting only the four loci with the highest variability values was found to offer an equivalent discriminatory index. Minimum spanning trees using the MIRU-VNTR data showed the hypothetical evolution of an apparent clonal group. MIRU-VNTR analysis was also applied to the isolates of 176 animals from 15 farms infected by M. bovis SB0121; in 10 farms, the analysis revealed the coexistence of two to five different MIRU types differing in one to six loci, which highlights the frequency of undetected heterogeneity.

A 16 kb naturally occurring genomic deletion including mce and PPE genes in Mycobacterium avium subspecies paratuberculosis isolates from goats with Johne's disease.

In this study we characterise the genomic and transcriptomic variability of a natural deletion strain of Mycobacterium avium subspecies paratuberculosis (MAP) prevalent in Spanish Guadarrama goats. Using a pan-genome microarray including MAP and M. avium subspecies hominissuis 104 genomes (MAPAC) we demonstrate the genotype to be MAP Type II with a single deletion of 19 contiguous ORFs (16 kb) including a complete mammalian cell entry (mce7_1) operon and adjacent proline-glutamic acid (PE)/proline-proline-glutamic acid (PPE) genes. A deletion specific PCR test was developed and a subsequent screening identified four goat herds infected with the variant strain. Each was located in central Spain and showed epidemiological links suggestive of transmission between herds. A majority of animals infected with the variant manifested a paucibacillary form of the disease. Comparisons between virulent complete genome compliment strains isolated from multibacillary diseased goats and the MAP variant strain during entry into activated macrophages demonstrated an increased sensitivity in the variant to intracellular killing in human and ovine macrophages. As PPE and mce genes are associated with mycobacterial virulence and pathogenesis we investigated the interplay of these gene sets during cell entry using the MAPAC array. This showed significant differential transcriptome profiles compared to full genome complement MAP controls that included changes in other undeleted mce operons and PE/PPE genes, esx-like signalling operons and stress response/fatty acid metabolism pathways. This strain represents the first report of a MAP Type II genotype with significant natural genomic deletions which remains able to cause disease and is transmissible in goats.

European 2--a clonal complex of Mycobacterium bovis dominant in the Iberian Peninsula.

Mycobacterium bovis isolates from the Iberian Peninsula are dominated by strains with spoligotype patterns deleted for spacer 21. Whole-genome sequencing of three Spanish strains with spacer 21 missing in their spoligotype pattern revealed a series of SNPs and subsequent screening of a selection of these SNPs identified one in gene guaA that is specific to these strains. This group of strains from the Iberian Peninsula missing spoligotype spacer 21 represents a new clonal complex of M. bovis, defined by the SNP profile with a distinct spoligotype signature. We have named this clonal complex European 2 (Eu2) and found that it was present at low frequency in both France and Italy and absent from the British Isles.

Application of the Enfer chemiluminescent multiplex ELISA system for the detection of Mycobacterium bovis infection in goats.

A study was conducted to optimise a multiplex serological immunoassay for use in identification of goats infected with Mycobacterium bovis. To assess assay specificity, 31 goats with a history of being free from M. bovis infection were used. To determine assay sensitivity, 180 Single Intradermal Comparative Tuberculin test (SICTT) positive goats were recruited. Additionally, 286 SICTT negative goats classed as potentially exposed animals present in the same positive herds were also included in the study. The results of the assay demonstrated a specificity of 100%. The multiplex assay detected 57/60 SICTT (95.0%) positive animals in one M. bovis infected herd and 120/120 (100%) in a second herd. In a separate experiment, 28 M. caprae culture confirmed infected goats from Spain were assayed, of which 24 (85.7%) were found positive in the test. The results show that inclusion of an antibody based assay can improve the ability to identify M. bovis and M. caprae infected goats. With further development and validation the multiplex assay may prove to be a useful tool for control of M. bovis and M. caprae infection in goats.

Tuberculosis in goats: assessment of current in vivo cell-mediated and antibody-based diagnostic assays.

Tuberculosis in goats, mainly caused by Mycobacterium bovis and M. caprae, is a zoonotic disease with implications for public health, as well as having an economic impact due to decreased goat production, increased mortality rates and costs of diagnosis. There is an increasing need for surveillance of tuberculosis-infected goat herds, particularly in countries that are not officially free of bovine tuberculosis, and goats sharing farms with cattle should be subjected to the official tuberculin test. In Spain, some regions have programmes for the control of tuberculosis in goats, applying the same diagnostic assays that are used for cattle. The objective of tuberculosis eradication in livestock requires adaptation of existing control strategies to include goats. As such, it is necessary to determine whether current diagnostic assays for tuberculosis in cattle will work as efficiently in the goat. This review provides an overview of current in vivo tools for diagnosis of caprine tuberculosis, including estimates of the sensitivity and specificity of tests performed in this species. The number of tested animals and co-infection with Mycobacterium avium subsp. paratuberculosis are also addressed, with the aim of demonstrating the limitations of current assays and the need for further research.

Progress in molecular typing of Mycobacterium avium subspecies paratuberculosis.

Mycobacterium avium subspecies paratuberculosis (M. a. paratuberculosis) is responsible for paratuberculosis or Johne's disease, a chronic inflammation of the gastrointestinal tract in different animal species. Some studies have also established a link between this microorganism and Crohn's disease in humans. Although, M. a. paratuberculosis is a difficult microorganism to cultivate in the laboratory (occasionally is non-cultivable), a proper molecular characterization of M. a. paratuberculosis is necessary to better understand the epidemiology of the disease, and design strategies to eradicate it. In the present review, we compile and discuss the recent progress attained in the diagnostic and characterization of this pathogen.